The aim of this research proposal is to elucidate the events in the infected cell concerned with the transcription and replication of the segmented influenza virus genome, and to determine the role of nucleus in these events. We intend to further characterize viral complementary RNA (cRNA), the product of transcription. Viral cRNA, the viral messenger RNA, is comprised of segments similar in size and number to the virion RNA (vRNA) segments. We will determine which cRNA segment codes for which virus-specific protein, and which cRNA segment is transcribed from which vRNA segment. Viral cRNA contains internal N6-methyladenosine (m6A) and a 5'-terminal 7-methylguan sine cap structure. Two different caps are present: one contains methylated adenosine, and the other methylated guanosine, as the penultimate nucleoside. We will determine the distribution of the two different caps, and of internal m6A, among the different cRNA segments. We will also determine whether the cRNA segments correspond to complete transcripts of the vRNA segments by sequence analysis of the 5' ends of cRNA and the 3' ends of vRNA. Specific probes, 125I-vRNA and 32P-complementary DNA, have enabled us to measure the number of cRNA and vRNA molecules in the nucleus and cytoplasm during infection. We have found that actinomycin D blocks the appearance in the cytoplasm of cRNA and not vRNA, whereas the cRNA resulting from primary transcription still appears in the nucleus. To identify the actinomycin D-sensitive nuclear step, we intend to further examine the appearance of cRNA during infection and to compare the characteristics (e.g., size, presence of poly A and 5' caps) of the cRNA synthesized in the presence and absence of actinomycin D. With these probes, we will also identify the defect(s) in RNA transcription and replication exhibited by temperature-sensitive viral mutants. We intend to develop an assay, employing hybridization and affinity chromatography, which will allow the detection of newly-synthesized cRNA and vRNA. With this assay, we will determine the intracellular site(s) of synthesis of cRNA and vRNA.